肺炎克雷伯菌菌毛的研究进展

肺炎克雷伯菌为条件致病菌,可引起肺炎、败血症等多种化脓性炎症,近年来肺炎克雷伯菌也成为医院内感染的主要致病菌之一。研究表明,菌毛作为细菌重要的毒力因子之一,在细菌黏附过程中起重要作用,细菌可借助于菌毛尖端黏附素黏附到宿主的组织器官,这是引起机体致病的首要条件。肺炎克雷伯菌菌毛包括Ⅰ型菌毛和Ⅲ型菌毛,绝大多数的肺炎克雷伯菌均可表达Ⅲ型菌毛,在医院感染的致病过程中起到关键作用。     

细菌粘附素的分子结构和装配机制

细菌感染的第一步是必须粘附于易感细胞,以获得立足点,在局部繁殖,释放毒素和酶类损坏组织,导致感染,细菌的粘附作用主要靠粘附素特异的识别结合到宿主细胞的受体,使细胞在局部定居,本文主要综述菌毛粘附素的分子结构及其因控制,菌毛粘附素如何通过四种典型途径装配,以及参与装配的蛋白质是如何协调功能的研究进展。     

细菌粘附宿主细胞外基质的分子基础

本文对细菌粘附宿主组织时与胞外基质（ＥＣＭ）结合的粘附素分子进行了综述。重点介绍了这类识别粘连基质分子的微生物表面成分的性质、结构和功能。具体涉及的金黄色葡萄球菌、化脓性链球菌、大肠杆菌、肺炎克雷伯菌、耶尔森菌和气单胞菌等,涉及的ＥＣＭ分子有纤维结合蛋白（Ｆｎ）、胶原及血纤维蛋白原。此外,还对ＭＳＣＲＡＭＭ－ＥＣＭ相互作用在细菌定居后,组织向性及致病中的作用进行了讨论。     

幽门螺杆菌粘附机制的研究进展

幽门螺杆粘附于胃黏膜是其进一步发挥致病作用的前提,本文从幽门螺杆菌的粘附素、粘附素相关受体、粘附于胃上皮细胞的病毒机制以及粘附素在粘附素在该菌疫苗构建中的作用对其粘附机制的研究进展作一综述。。     

肺炎链球菌粘附机制的研究现状

肺炎链球菌粘附宿主我肺炎链球菌侵袭、感染宿主细胞的先决条件。粘附过程是特异的,是细菌表面的粘附分子和宿主细胞膜受体相互作用的结果。英膜对肺炎链球菌的粘附无影响,而细胞壁（ＣＷ）在介导肺炎链附粘附宿主细胞过程中起重要作用;ＣＷ亚组人脂磷酸（ＬＴＡ）介导肺炎链球菌的粘附过程,并导致炎症反应;细菌表面的结构蛋白或分泌蛋白是细菌与宿主细胞连接的桥梁;肺炎链球菌能与宿主细胞外基质蛋白特异性结合,进而粘附宿主     

白色念珠菌粘附的研究进展

本文论述了白色念珠菌（简称白念菌）粘附的分子机理和实验模型观察,从病原菌和宿主两方面探讨了白念菌感染的发病机理,并从阻断白念菌对宿主细胞的粘附着手,展望防治白念菌感染的新途径和措施。     

嗜酸乳杆菌NCFM粘附宿主后对其粘附相关基因表达的影响

摘要：【目的】益生菌粘附于肠道上皮细胞上是它的一种益生作用。本研究通过体内外实验,分析嗜酸乳杆菌NCFM对粘附相关基因的影响。【方法】利用GO (Gene Ontolog) 分类筛选Human Genome U133 Plus 2.0 Array基因表达谱芯片中的粘附相关基因,通过体外Caco-2细胞培养模型和体内小鼠粘附模型,采用Real-time PCR方法对粘附相关基因进行验证分析。【结果】经NCFM作用后,12个粘附相关基因呈上调表达。利用Real-time PCR验证,12个基因在体内和体外经嗜酸乳杆菌NCFM作用后亦均同样为上调表达,其中CCL2基因上调表达最为明显。【结论】经体内外研究表明,嗜酸乳杆菌NCFM粘附肠上皮细胞后能够引起宿主粘附相关基因出现特定表达变化,为今后深入揭示其粘附作用提供必要基础。     

clpE基因缺失对肺炎链球菌毒力的影响

【目的】 探索clpE基因缺失对肺炎链球菌毒力的影响。【方法】 用长臂同源多聚酶链式反应(LFH-PCR)方法失活clpE基因,用PCR、测序鉴定缺失菌株,通过动物实验观察clpE基因缺失株毒力改变情况, 同时用细胞实验比较clpE基因缺失株和野生菌对宿主细胞的粘附和侵袭能力,最后用实时荧光定量PCR分析自溶素(major autolysin A,lytA)、表面黏附素A(pneumococcal surface adhesion A,psaA)、溶血素(pneumolysin,ply)、肺炎球菌表面蛋白A（pneumococcal surface protein A, pspA)和神经氨酸酶（neuraminidase, nanA）的表达。 【结果】小鼠毒力实验表明野生菌株半数致死时间54h,而缺失株半数致死时间为21d,两者比较有统计学差异（P<0 .0l）;缺失菌在对宿主细胞的粘附能力明显低于野生菌株（P<0.05）。实时荧光定量PCR显示clpE缺失株的五个毒力因子mRNA表达水平均低于野生菌,两者比较有统计学差异（P<0. 05）;【结论】ClpE通过调控肺炎链球菌多种毒力因子表达,而影响其毒力。     

绿脓杆菌的粘附素

绿脓杆菌的粘附功能被认为是绿脓杆菌呼吸道感染的首要一步。粘液型菌株是由细胞外粘多糖（ＭＥＰ）介导,而非粘液型菌株则由菌毛（Ｐｉｌｉ）介导,国内外文献早有报道并认为ＭＥＰ和菌毛均为绿脓杆菌的粘附素。绿脓杆菌的菌毛与菌体的粘附、动力和噬菌体的吸附有关。ＭＥＰ除了粘附功能外,尚与抑制白细胞的吞噬作用、抑制抗ＬＰＳ抗体介导的调理吞噬作用有关,并能增强菌株对抗体包被的抵抗力。     

沙门菌噬菌体抗性菌的筛选鉴定及致病力研究

【目的】筛选鉴定沙门菌噬菌体侵染裂解过程中的抗性菌株,研究抗性菌株的生物学特性及致病力的差异,为解决噬菌体治疗应用中的抗性菌问题提供理论依据。【方法】本研究通过次级感染法和双层平板法筛选沙门菌噬菌体抗性菌,通过生物学特性和毒力基因检测比较宿主菌ATCC 13076及其噬菌体抗性菌株R3之间的差异,并通过小鼠攻毒实验和细胞粘附实验比较致病力强弱。【结果】噬菌体抗性菌株R3的生长速度较宿主菌略慢;生化及毒力基因检测均表明抗性菌株与宿主菌无差异;与宿主菌相比,抗性菌R3的LD50增加了74.8%(P0.05);对MODE-K细胞粘附能力稍弱,但是差异不显著。【结论】该研究表明,与噬菌体宿主菌相比,噬菌体抗性菌株的生物学特性和毒力基因并没有改变,对小鼠致病力减弱,但是对MODE-K细胞粘附能力差异不显著。     

Characterization of the type 3 fimbriae with different MrkD adhesins: possible role of the MrkD containing an RGD motif

Four novel mrkD alleles namely mrkD(V1), mrkD(V2), mrkD(V3), and mrkD(V4) were identified in seventeen Klebsiella pneumoniae meningitis strains using PCR-RFLP and sequence determination. Comparative analysis revealed a most variable region containing an RGD motif in the receptor domain of MrkD(V3). In order to determine if the sequence confers the K. pneumoniae mrkD(V3) the highest level of the fimbrial activity, a type 3 fimbriae display system was constructed in Escherichia coli. The E. coli JM109[pmrkABCD(V3)F] displaying meshwork-like fimbriae also had the most fimbrial activity, supporting a possible role of the varied sequences. In a dose-dependent manner, the GRGDSP hexapeptide appeared to inhibit the adhesion of the E. coli JM109[pmrkABCD(V3)F] to HCT-8, an ileocecal epithelial cell line. In addition, the adhesion activity was reduced by the addition of anti-alpha5beta1 integrin monoclonal antibody, indicating that the RGD containing region in MrkD(V3) is responsible for the binding of type 3 fimbriae to integrin.     

Structural and Population Characterization of MrkD,the Adhesive Subunit of Type 3 Fimbriae

Type 3 fimbriae are adhesive organelles found in enterobacterial pathogens. The fimbriae promote biofilm formation on biotic and abiotic surfaces; however, the exact identity of the receptor for the type 3 fimbriae adhesin, MrkD, remains elusive. We analyzed naturally occurring structural and functional variabilities of the MrkD adhesin from Klebsiella pneumoniae and Escherichia coli isolates of diverse origins. We identified a total of 33 allelic variants of mrkD among 90 K. pneumoniae isolates and 10 allelic variants among 608 E. coli isolates, encoding 11 and 9 protein variants, respectively. Based on the level of accumulated silent variability between the alleles, mrkD was acquired a relatively long time ago in K. pneumoniae but recently in E. coli. However, unlike K. pneumoniae, mrkD in E. coli is actively evolving under a strong positive selection by accumulation of mutations, often targeting the same positions in the protein. Several naturally occurring MrkD protein variants from E. coli were found to be significantly less adherent when tested in a mannan-binding assay and showed reduced biofilm-forming capacity. Functional examination of the MrkD adhesin in flow chamber experiments determined that it interacts with Saccharomyces cerevisiae cells in a shear-dependent manner, i.e., the binding is catch-bond-like and enhanced under increasing shear conditions. Homology modeling strongly suggested that MrkD has a two-domain structure, comprising a pilin domain anchoring the adhesin to the fimbrial shaft and a lectin domain containing the binding pocket; this is similar to structures found in other catch-bond-forming fimbrial adhesins in enterobacteria.     

Adherence epitopes of Mycoplasma genitalium adhesin.

The adherence-mediating sites of the 153 kDa adhesin of Mycoplasma genitalium (MgPa-protein) were characterized at the amino acid sequence level using six monoclonal anti-MgPa antibodies which showed adherence-inhibiting activity. For characterization of the regions to which antibody bound, three segments of the adhesin (N-terminal region, a D1-domain located approximately in the middle of the molecule and a D2-domain located near to the C-terminus) were synthesized as overlapping octapeptides. These regions were chosen in analogy to the three domains of Mycoplasma pneumoniae that are involved in the adhesion process. Whereas two monoclonal antibodies (mAb 5B11 and mAb 6F3) bound exclusively to an epitope in the N-region, mAb 3B7 and mAb 6A2 reacted with two distinct epitopes of the D2-domain only. Binding to short synthetic peptides of different regions was analysed for mAb 3A12 (N-region and D1-region) and mAb 2B6 (N-region and D2-region). Close proximity of the N-region and the D2-region in the native MgPa-protein of M. genitalium was indicated in a competitive ELISA test, using freshly harvested M. genitalium cells. Epitope mapping and competition experiments with monoclonal anti-MgPa antibodies revealed interesting differences in the adherence-mediating sites of MgPa and the adhesin (P1-protein) of M. pneumoniae. Whereas a three-dimensional arrangement of protein loops is suggested for both native adhesins, the MgPa-protein and the P1-protein adherence-mediating epitopes are located in non-homologous regions of these two related proteins.(ABSTRACT TRUNCATED AT 250 WORDS)     

Occurrence and characteristic of P-like adhesin among Klebsiella strains

Colonisation and remaining of microorganism on mucus membrane of microorganism is tightly connected with adhesion mechanisms and determine the first step of physiological settlement of the organism or the first stage of clinically demonstrated infection. In Klebsiella rods there are known three types of fimbrial adhesins (type 1, 3 and KPF-28) and non-fimbrial adhesin CF29K. It is stated that Klebsiella strains adhesions are responsible for their adherence to the epithelial cells of both respiratory and urinary tracts and to intestine epithelium. The in vitro research affirmed Klebsiella rods adherence to protein matrix. The aim of our work was the establishment of character, receptor specificity and the appearance frequency of P-like called adhesin. The frequency of expression of P-like adhesin was estimated among 380 isolated from the patients strains on the basis of agglutinating methods. The amorphic character of P-like adhesin was proved using electron microscopy method. The isolation and purification of P-like protein with a help of affinity chromatography enabled to estimate the receptor specificity of the adhesin. The receptor specificity was established as similar to E.coli PapG adhesin.     

异源表达木糖异构酶基因对克雷伯氏菌合成1,3-丙二醇的影响

【目的】提高克雷伯氏菌胞内还原力以强化1,3-丙二醇合成。【方法】将来源于大肠杆菌的木糖异构酶基因在克雷伯氏菌中异源表达,构建重组菌。研究重组菌添加不同浓度木糖为辅底物与甘油共发酵过程中代谢产物和NADH的变化规律。【结果】与对照菌相比,重组菌细胞内还原力NADH提高了0.1?0.3倍,1,3-丙二醇产量达到23.31 g/L,提高20%,1,3-丙二醇转化率从0.60 mol/mol提高到0.73 mol/mol。【结论】木糖异构酶基因的表达强化了木糖代谢途径,经磷酸戊糖途径积累大量还原力,促进了1,3-丙二醇的生成。     

超广谱β-内酰胺酶肺炎克雷伯菌噬菌体F20的分离鉴定及其对小鼠败血症治疗效果

【背景】肺炎克雷伯菌是引起临床感染的重要条件致病菌之一,肺炎克雷伯菌中产超广谱β-内酰胺酶(Extended-spectrum beta-lactamases,ESBLs)的耐药菌株增多迫切需要找到一种新的治疗方法。【目的】自污水中分离超广谱β-内酰胺酶肺炎克雷伯菌噬菌体,并明确其生物学特性、观察其治疗小鼠产ESBLs肺炎克雷伯菌感染的疗效。【方法】电镜观察F20形态,调查其噬菌谱、生长曲线等生物学特性。建立小鼠败血症感染模型观察F20治疗小鼠肺炎克雷伯菌感染的疗效。【结果】F20在其宿主菌的菌苔上形成裂解性噬菌体所具有的完全透明的噬菌斑,电镜观察F20具典型的有尾噬菌体目长尾病毒科病毒的形态特征。一步生长曲线显示F20的潜伏期为18 min,裂解量为89 PFU/细胞。稳定性试验显示F20在pH 5.0-9.0及50°C环境均具良好稳定性。使用噬菌体F20对败血症小鼠治疗后,治疗组小鼠各外周血和各脏器(肺脏、肝脏、脾脏和肾脏)中的细菌数也显著小于对照组细菌数(P0.001),与对照组相比下降大约1–3数量级。F20治疗败血症小鼠存活率达到87.5%,无毒副作用,而对照组小鼠在1 d内全部死亡,可显著提高小鼠的存活率(P0.001)。【结论】新分离的裂解性噬菌体F20在小鼠体内能安全有效地治疗超广谱β-内酰胺酶肺炎克雷伯菌引起的败血症,可作为生物抗菌剂的有效成分。     

Purification and characterization of an adhesin from Pasteurella haemolytica

We purified an adhesin from Pasteurella. haemolytica by affinity chromatography using glutaraldehyde treated rabbit erythrocytes stroma. The adhesin is a protein of 68 kDa, as determined by SDS-PAGE, and the most abundant amino acids constituting this protein were Gly, Ser, Glx, and Ala, and low concentrations of Cys, Met, and Tyr residues were also found. The N-terminal sequence of the adhesin is ANEVNVYIYKQPYLI. No carbohydrate residues were detected. The adhesin agglutinated rabbit erythrocytes but when the latter were desialylated or pronase treated the agglutinating activity was abolished. The agglutinating activity of the adhesin was inhibited with N-acetyl-D-glucosamine (GlcNAc), and in a lesser degree with N-acetyl-neuraminic acid (NeuAc). GalNAc, N-glycolyl-neuraminic acid, N-deacetylated GlcNAc, or neutral sugars do not modify the activity of the adhesin. The equatorial -OH on C4 and the NH-acetylated group on C2 from GlcNAc, as well as the 4-OH and NH-acetylated group on C5 from NeuAc seem to be responsible for the interaction with the adhesin. The protein is divalent cation-dependent and thermolabile. As for the agglutinating activity, the adhesion of P.haemolytica to tracheal cell-cultures was inhibited by GlcNAc, NeuAc or the purified adhesin, strongly suggesting that the P.haemolytica adhesin plays an important role in infection.     

一株新型裂解K63荚膜型肺炎克雷伯菌的噬菌体分离鉴定和生物学特性研究及全基因组分析

[背景]噬菌体具有特定的杀菌能力,对生态和细菌的进化具有重要影响。近年来由于多重耐药细菌的全球出现,噬菌体疗法逐渐引起了人们的关注。[目的]对一株新型裂解K63荚膜型肺炎克雷伯菌的噬菌体vB_KpnP_IME308进行生物学特性研究、测序和比较基因组学的分析。[方法]以一株从临床分离到的肺炎克雷伯菌为宿主菌分离噬菌体,应用双层平板法进行噬菌体最佳感染复数(optimal multiplicity of infection)、一步生长曲线(one-step growth curve)、温度以及pH敏感性实验测定,纯化噬菌体并通过透射电镜观察噬菌体形态;应用标准的苯酚-氯仿提取方案提取噬菌体全基因组,使用Illumina MiSeq测序平台进行噬菌体全基因组测序,测序后对噬菌体全基因组序列进行组装、注释、进化和比较基因组学分析。[结果]分离到一株新型的肺炎克雷伯菌噬菌体,命名为vB_KpnP_IME308;其最佳感染复数为0.001,一步生长曲线结果显示,其感染宿主菌的潜伏期约为20 min,裂解期约为80 min,平均裂解量330PFU/cell;噬菌体vB_KpnP_IME308在4-50℃和pH 5.0-10.0范围内稳定;电镜观察该噬菌体属于短尾噬菌体科(Podoviridae)。基因组测序结果表明,噬菌体基因组全长为43 091bp,(G+C)mol%含量为53.9%,(A+T)mol%含量为46.1%。BLASTn比对结果表明,该噬菌体与目前已知噬菌体基因组仅84%区域有相似性。噬菌体进化树结果表明该噬菌体属于Autographivirinae亚科的Drulisvirus属的成员。[结论]从医院污水中分离鉴定了一株新型的肺炎克雷伯菌噬菌体,表征并分析了噬菌体全基因组序列,这些结果均表明该噬菌体具有开发为抗肺炎克雷伯菌制剂的潜力,为噬菌体治疗多重耐药细菌感染奠定了基础。     

Surfactant protein D binds selectively to Klebsiella pneumoniae lipopolysaccharides containing mannose-rich O-antigens

Surfactant protein D (SP-D) plays important roles in the regulation of innate immune responses in the lung. We have previously shown that SP-D can agglutinate and enhance the macrophage-dependent killing of specific unencapsulated phase variants of Klebsiella pneumoniae. In the present studies, we used 16 clinical isolates of Klebsiella representing four O-serotypes and examined the interaction of SP-D with their isolated LPSs. Although SP-D bound to the core oligosaccharide of rough LPS from all isolates, it selectively bound to smooth forms of LPS expressed by O-serotypes with mannose-rich repeating units in their O-polysaccharides. SP-D was more potent in agglutinating unencapsulated phase variants of O-serotypes expressing these SP-D "reactive" O-polysaccharides, and more effectively inhibited the adhesion of these serotypes to lung epithelial cells. This novel anti-adhesion activity required the multimerization of trimeric SP-D subunits (dodecamers). Klebsiella serotypes expressing "nonreactive" LPS O-Ags were isolated at a significantly higher frequency from patients with K. pneumoniae. Our findings suggest that SP-D plays important roles in the clearance of opportunistic Gram-negative bacteria and contributes to known serotypic differences in the pathogenicity of Klebsiella through specific interactions with O-polysaccharides.